Inhibition of Schwann Cell Pyroptosis Promotes Nerve Regeneration in Peripheral Nerve Injury in Rats

Background Peripheral nerve injury (PNI) is one of the most debilitating injuries, but therapies for PNI are still far from satisfactory. Pyroptosis, a recently identified form of cell death, has been demonstrated to participate in different diseases. However, the role of pyroptosis of Schwann cells in PNI remains unclear. Methods We established a rat PNI model, and western blotting, transmission electron microscopy, and immunofluorescence staining were used to confirm pyroptosis of Schwann cells in PNI in vivo. In vitro, pyroptosis of Schwann cells was induced by lipopolysaccharides (LPS)+adenosine triphosphate disodium (ATP). An irreversible inhibitor of pyroptosis, acetyl (Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), was used to attenuate Schwann cell pyroptosis. Moreover, the influence of pyroptotic Schwann cells on the function of dorsal root ganglion neurons (DRGns) was analyzed by a coculture system. Finally, the rat PNI model was intraperitoneally treated with Ac-YVAD-cmk to observe the effect of pyroptosis on nerve regeneration and motor function. Results Schwann cell pyroptosis was notably observed in the injured sciatic nerve. LPS+ATP treatment effectively induced Schwann cell pyroptosis, which was largely attenuated by Ac-YVAD-cmk. Additionally, pyroptotic Schwann cells inhibited the function of DRGns by secreting inflammatory factors. A decrease in pyroptosis in Schwann cells promoted regeneration of the sciatic nerve and recovery of motor function in rats. Conclusion Given the role of Schwann cell pyroptosis in PNI progression, inhibition of Schwann cell pyroptosis might be a potential therapeutic strategy for PNI in the future.


Introduction
Peripheral nerve injury (PNI) is a common clinical problem that results in substantial disability worldwide [1]. Statistics show that the incidence of PNI ranges from 1.3 to 2.3 per 100,000 people in developed countries, and this incidence may be higher in developing countries [2]. The function of the peripheral nervous system can recover to some extent after PNI, and the extent depends on the severity of the injury and the method of treatment; thus, it is different from the central nervous system [3]. The regenerative process of peripheral nerves is complicated, and the effect of nerve regeneration is always unsatisfactory. The limited treatments for PNI have placed a major burden on social development, yet its underlying mechanisms remain largely unknown.
Schwann cells are a kind of major glial cell in peripheral nervous systems. These cells play a critical role in the regeneration of peripheral nerves, such as the repair of damaged neurons after PNI. After peripheral nerve injury, axons on the distal side of the injured nerve quickly degenerate and disintegrate, and then, debris is phagocytosed and cleared by Schwann cells and macrophages; these phenomena are called Wallerian degeneration [4,5]. Schwann cells are involved in Wallerian degeneration and other processes of peripheral nerve regeneration [6]. These cells can secrete neurotrophic factors, promote the survival of damaged neurons, and participate in the formation of nerve fibers in the peripheral nervous system. Moreover, Schwann cells can initiate the immune response to recruit monocytes and macrophages to clear myelin debris in the injured sciatic nerve [7]. Therefore, it is important to further explore the roles of Schwann cells in peripheral nerve regeneration.
Pyroptosis is a recently discovered form of programmed lytic cell death. This process is induced by a series of physiological or pathological stimuli, such as glucocorticoids, hypoxia [8], DNA damage [9], and viral infection [10]. These stimuli result in mitochondrial damage and an increase in reactive oxygen species (ROS) [11]. In the latter, NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammatory bodies interact with apoptosisassociated speck-like protein containing CARD (ASC) and the effector molecule cysteinyl aspartate-specific protease-1 precursor (pro-caspase-1), resulting in the formation of inflammasome complexes and Cleaved-casp-1 [12,13]. Gasdermin D (GSDMD) and the precursor inflammatory factors pro-IL-18 and pro-IL-1β were then cleaved by Cleaved-casp-1, the pivotal mediating factor for pyroptosis. GSDMD is composed of the N-terminal fragment of GSDMD (N-GSDMD) and the C-terminal fragment of GSDMD (C-GSDMD). After cleavage by Cleaved-casp-1, N-GSDMD oligomerizes in the cytomembrane to form pores that increase cell membrane permeability. Subsequently, IL-18 and IL-1β are released from the pores in the cytomembrane generated by N-GSDMD [14]. Recent studies have verified that pyroptosis is involved in the pathological process of many diseases, such as septicemia [15], diabetic cardiomyopathy [16], atherosclerosis [17,18], various tissue injuries [19,20], and even Alzheimer's disease [21]. Moreover, researchers have found that macrophage pyroptosis is involved in PNI in mice [22]. However, the role of Schwann cell pyroptosis in PNI is still unknown.
In this study, we explored the role of Schwann cell pyroptosis in PNI. We investigated the ability of LPS+ATP to induce Schwann cell pyroptosis and Ac-YVAD-cmk to suppress pyroptosis. Then, we explored the effect of Schwann cell pyroptosis on the function of surrounding neurons. Moreover, the effect of Ac-YVAD-cmk on peripheral nerve regeneration was observed in vivo.

Ethics Statement and Animals.
Six-to eight-week-old male Sprague-Dawley rats (200-250 g) were housed in temperature-and humidity-controlled quarters in the Lab Animal Center of Zhongshan Hospital, Fudan University. The study was approved by the ethics committee of Zhongshan Hospital, and all experimental procedures were approved by the Animal Care and Use Committee of Zhongshan Hospital.

Animal Experiments.
Animal experiment was divided into two parts in the study; the total number of rats was 180. We first confirmed Schwann pyroptosis exist in PNI, the total number of rats was 126, and the rats were divided into seven groups (n = 18). Then, the rat PNI model was intraperitoneally treated with Ac-YVAD-cmk to observe the effect of pyroptosis on nerve regeneration, the total number of rats was 54, and the rats were divided into three groups (n = 18).

PNI Model.
Rats were randomly divided into groups as follows: sham-operation controls (control, n = 18) and the PNI group (n = 108) at 0.5, 1, 2, 3, 7, and 14 d (n = 18 for each observation time point). All rats were anesthetized and kept on a heating pad during anesthesia. The left hind leg of each rat was prepared for surgery after shaving and disinfection. With aseptic techniques, the sciatic nerve was isolated and transected in a transverse incision below the femur in the PNI group, and then, the proximal and distal nerve stumps were sutured under a microscope [23]. The sciatic nerve was exposed without damage, and then, the muscle layers and skin were sutured in the control group. The nerve samples 1 cm distal to the nerve transection site were collected separately from rats after transection in the PNI group, and nerve samples of equal length were also collected in the control group. All the procedures were performed by the same investigator.

Regeneration of Peripheral
Nerve. The rat PNI model was intraperitoneally treated with Ac-YVAD-cmk to observe the effect of pyroptosis on nerve regeneration. A total of 54 rats were randomly divided into three groups as follows: Group 1: sham operation (control, n = 18); Group 2: sciatic nerve transection with immediate repair followed by intraperitoneal administration of placebo (PNI, n = 18); Group 3: sciatic nerve transection with immediate repair followed by intraperitoneal administration of Ac-YVAD-cmk (PNI +AC, n = 18). Ac-YVAD-cmk (25 mg/kg) or an equal volume of placebo was administered daily for 7 days after surgery [24,25]. Sciatic nerve segments 1 cm distal to the nerve transected site were taken on the 7th postoperative day for western blotting and immunofluorescence staining analysis and on the 90th postoperative day for transmission electron microscope analysis. All the procedures were performed by the same investigator.

Western
Blotting. Total protein was extracted from sciatic nerve samples, cultured Schwann cells or using RIPA buffer (Lot: #P0013B, Beyotime). Twenty-five milligrams of protein was resolved by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were incubated with primary antibodies overnight at 4°C. Then, the cells were incubated with secondary antibodies for 1 h at room temperature. Finally, proteins were visualized using a chemiluminescent peroxidase substrate.

Transmission Electron Microscopy.
Transmission electron microscopy was used to verify the morphology of pyroptotic Schwann cells in the injured sciatic nerve. The balllike bulge morphology of pyroptosis in the injured sciatic nerve was observed by transmission electron microscopy at different time points after injury, and representative images are shown in this study.
In the Ac-YVAD-cmk treatment experimental section in vivo, the electron micrographs from six random fields (×1000 magnification) of each group were observed, and the average myelinated axon diameter and thickness of the myelin sheath were assessed based on the electron micrographs by using ImageJ software [30,31].
2.12. Coculture System. Transwell chamber systems were constructed to investigate the effect of pyroptotic Schwann cells on the function of DRGns [32]. Schwann cells were seeded in the upper chamber, and DRGns were seeded in the lower chamber. DRGns were cultured in a 24-well plate for 1 week before the transwell cell insert (0.4 μm pore size, Corning) containing Schwann cells (1 × 10 4 ) that were treated with LPS+ATP+PBS, LPS+ATP+Ac-YVAD-cmk (AC), or LPS+ATP+neutralizing antibody (NA) was added to each well of the plate. The control group is the DRGns cocultured with Schwann cells without any treatment. The transwell cell insert was washed three times with PBS before being added to the well of the plate to eliminate the effect of residual ATP on inferior ventricular DRGns. After 24 h of coculture, DRGns were collected for immunofluorescence staining or PCR assays. For immunofluorescence staining, the average body size and axonal length of DRGns were analyzed using Image-Pro-Plus (SIS, Münster, Germany).
2.13. Walking Track Analysis. The sciatic functional index (SFI) is generally used to assess behavioral recovery and restoration of function after sciatic nerve injury [33]. SFI was performed to assess sciatic behavioral function at different time points after surgery in this study. Briefly, hind paws were dipped in black ink, and rats of different groups were allowed to walk down a narrow channel, making footprints 3 Mediators of Inflammation on a white paper. SFI was calculated according to the following equation: SFI = −38:3 × ðEPL − NPLÞ/NPL + 109:5 × ð ETS − NTSÞ/NTS + 13:3 × ðEIT − NITÞ/NIT − 8:8. PL is the distance from the heel to the third toe, TS is the distance from the first to the fifth toe, and IT is the distance from the second to the fourth toe. E and N represent the experimental and normal groups, respectively.
2.14. Statistical Analysis. Statistical analyses were analyzed using GraphPad Prism 8.0 software. One-way ANOVA was used to evaluate the differences among the different groups. Statistical analyses were performed with SPSS 23.0 software (SPSS, Inc., Chicago, IL, USA). p < 0:05 was considered statistically significant.

PNI Induces Schwann Cell Pyroptosis In Vivo.
We first verified whether pyroptosis exists in Schwann cells in the PNI rat model using western blotting, immunofluorescence staining, and transmission electron microscopy. The levels of pyroptosis-associated proteins in the sciatic nerve were evaluated by western blotting after PNI at different time points after surgery. Compared with those of the control group, the expression levels of N-GSDMD and Cleavedcasp-1 were notably upregulated in transected rat sciatic nerve tissue and peaked on the 3rd day in the PNI model (Figures 1(a)-1(c)). In addition, GSDMD colocalization with S100 peaked on the 3rd day after PNI (Figure 1(d)), which was consistent with the western blot results, suggesting that high levels of GSDMD were expressed in Schwann cells on the 3rd postoperative day. Furthermore, ball-like bulges were observed in damaged Schwann cells, especially on the 3rd day (Figures 1(e) and 1(f)). These results suggested that PNI induced Schwann cell pyroptosis in rats.

LPS+ATP Induce Pyroptosis in Schwann Cells.
To further confirm the phenotype of pyroptosis in Schwann cells, we treated these cells with different concentrations of LPS (0.1, 1 μg/ml) and ATP (2.5, 5 mM) to induce pyroptosis, except for the control group. Previous studies revealed that the inflammatory response was linked to pyroptosis [34]; thus, we wondered whether combining LPS with ATP induced an inflammatory response in Schwann cells. The ELISA results revealed that the inflammatory cytokines IL-18 and IL-1β in the culture supernatant increased and peaked in the 1 μg/ml LPS+5 mM ATP (6 h) group after LPS+ATP treatment (Figures 2(a) and 2(b)). Moreover, the western blotting results demonstrated that the protein levels of NLRP3, N-GSDMD, and Cleaved-casp-1 were significantly elevated and peaked in the 1 μg/ml LPS+5 mM ATP (6 h) group (Figures 2(c)-2(f)) compared to the control group. Herein, the above data suggested that combining LPS with ATP could induce pyroptosis in Schwann cells, and a 1 μg/ml LPS+5 mM ATP (6 h) model was used to induce pyroptosis in subsequent experiments.
3.3. Ac-YVAD-cmk (AC) Attenuates Pyroptosis Induced by LPS+ATP in Schwann Cells. To further confirm pyroptosis in Schwann cells, we used the caspase-1 inhibitor Ac-YVAD-cmk to attenuate LPS+ATP-induced pyroptosis in Schwann cells [35]. The CCK-8 assay showed that the cell viability of the 50 μM AC/LPS+ATP group was higher at 12, 24, and 48 h than that of the LPS+ATP group, while it was still lower than that of the control group (p < 0:05, Figure 3(a)). We also found that the levels of IL-18 and IL-1β in the 50 μM AC/LPS+ATP group were obviously downregulated (p < 0:05, Figures 3(b) and 3(c)). Furthermore, the protein levels of NLRP3, N-GSDMD, and Cleaved-casp-1 were significantly reduced compared with those in the LPS +ATP group (Figures 3(d)-3(g)). Finally, GSDMD was colocalized with DiI, a cell membrane fluorescent probe (Figure 3(h)). The levels of GSDMD in the LPS+ATP group were significantly higher than those in the control group in both the cytoplasm and membrane of Schwann cells. The mean fluorescence intensity (MFI) of GSDMD at the surface and cytoplasm was decreased in the LPS+ATP+50 AC group compared to the LPS+ATP group. According to the above results, 50 μM Ac-YVAD-cmk was used to inhibit pyroptosis in the following study.

Pyroptosis of Schwann Cells Inhibits the Function of
Cocultured DRGns. Schwann cells and DRGns were cocultured using the transwell system (Figure 4(a)). The representative morphology of primary DRGns before coculture with Schwann cells on the 7th day after extraction is shown at different magnifications (Figures 4(b) and 4(c)). Real-time PCR results showed that the gene expression of IL-6 in DRGns in the LPS+ATP+PBS group and the LPS+ATP+NA group was significantly higher than that in the control group (p < 0:05), and the gene expression of IL-6 in DRGns in the LPS+ATP +AC group and the LPS+ATP+NA group was significantly lower than that in the LPS+ATP+PBS group (Figure 4(d)). There was no significant difference in the expression of TNF-α (Figure 4(e)). Immunofluorescence staining was used to detect the location of specific markers of neurons, βIII-Tubulin and NeuN, of DRGns ( Figure 4(f)) to evaluate the function of DRGns. The MFI of βIII-Tubulin and NeuN in the LPS+ATP+PBS group was significantly lower than that in the LPS+ATP+AC group and the LPS+ATP+NA group (p < 0:05), and there was no significant difference between the control group and the LPS+ATP+AC group. The average body size and axonal length of DRGns cocultured with Schwann cells from different groups were analyzed. The average body size and axonal length of DRGns in the LPS +ATP+PBS group were significantly lower than those in the three other groups, and there was no significant difference between the control group and the LPS+ATP+AC group (Figures 4(g)-4(h)). Taken together, these data proved that pyroptosis of Schwann cells inhibited the function of cocultured DRGns and that Ac-YVAD-cmk could attenuate the inhibitory effects of pyroptotic Schwann cells on cocultured DRGns.       - -  Mediators of Inflammation PNI+AC group compared with the PNI group (p < 0:05).
These aforementioned results proved that Ac-YVAD-cmk could suppress injury-induced Schwann cell pyroptosis in the sciatic nerve. Transmission electron microscopy was used to observe the morphological features of peripheral nerves in different groups on the 90th postoperative day (Figure 5(f)). The average myelinated axon diameter and thickness of the myelin sheath of regenerated nerve fibers in the PNI+AC group were significantly better than those in the PNI group, while they were still lower than those in the control group (p < 0:05, Figures 5(g) and 5(h)).
To assess the possible effects of Ac-YVAD-cmk on motor functional recovery, we determined the SFI to evaluate the recovery of motor function after sciatic nerve injury in rats. The SFI value varies from −100 to 0, with −100 and 0 indicating absolute loss and normal motor function of the sciatic nerve, respectively. No significant difference in the SFI value was observed among rats in different groups on the 1st, 15th, and 30th postoperative day, but the SFI value was significantly higher in the rats treated with Ac-YVAD-cmk at other time points after surgery than in the rats treated with placebo (p < 0:05, Figure 5

Discussion
In this study, we established a peripheral nerve injury model and confirmed that pyroptosis is a major type of Schwann cell death. We confirmed that LPS+ATP treatment could effectively induce pyroptosis of Schwann cells, which could be attenuated by the pyroptosis inhibitor Ac-YVAD-cmk. Pyroptosis of Schwann cells can lead to dysfunction of DRGns by the inflammatory mediators IL-1β and IL-18. Most importantly, Ac-YVAD-cmk was shown to promote nerve regeneration in peripheral nerve injury by attenuating pyroptosis of Schwann cells in rats.
Complete transection injury is usually used as a model for the study of PNI [22], and we used transection injuries as a model in this study. In the present study, transection injury released many danger-associated molecular patterns (DAMPs) [36], which induced subsequent Schwann cell pyroptosis. Herein, pyroptotic Schwann cells were found in the PNI rat model, especially on the 3rd postoperative day. Pyroptosis of Schwann cells was attenuated on the 7th and 14th postoperative day. We speculated that many DAMPs were gradually released from damaged tissues and accumulated at the surrounding Schwann cells, and these cytokines  Mediators of Inflammation and mediators induced Schwann cell pyroptosis [37]. Then, the number of pyroptotic Schwann cells was reduced along with the repair of tissue damage [36]. Thus, these data suggested that DAMPs induced Schwann cell pyroptosis. LPS+ATP treatment is commonly used to induce cell pyroptosis in vitro [38][39][40][41]. Consistent with previous findings, LPS combined with ATP elevated the levels of the pyroptosisassociated proteins NLRP3, N-GSDMD, and Cleaved-casp-1. The caspase-1 inhibitor Ac-YVAD-cmk dramatically inhibited pyroptosis induced by LPS+ATP in Schwann cells. These data revealed that pyroptosis in the Schwann cell model was established using LPS+ATP treatment.
Schwann cells have positive effects on the function of neuronal cells [42,43]; however, few studies have reported the effects of pyroptotic Schwann cells. Herein, we found that pyroptotic Schwann cells suppressed the function of neurons and promoted the inflammatory response. However, inhibition of pyroptosis in Schwann cells improved neuron function and attenuated inflammatory mediator production in peripheral nerves. Pyroptosis of cells can release many inflammatory mediators, such as IL-1β and IL-18, and these mediators further induce pyroptosis in the surrounding cells, which ultimately exacerbates tissue damage. Previous studies proved that IL-1β and IL-18 induced nerve injury and impaired the  [42,43]. Our study revealed that Schwann cell pyroptosis released IL-1β and IL-18. Further analysis showed that downregulation of inflammatory cytokines using a neutralizing antibody also improved the function of DRGns. Together, these data suggest that pyroptosis of Schwann cells causes dysfunction of dorsal root ganglion neurons through the release of inflammatory cytokines.
Ac-YVAD-cmk is usually used to inhibit pyroptosis by intraperitoneal injection in vivo [35,44]. Next, we validated the effect of Ac-YVAD-cmk in the regeneration of peripheral nerves. Levels of pyroptosis-associated proteins were found to be dramatically downregulated in the PNI +AC group compared with the PNI group, which proves that Ac-YVAD-cmk is effective in inhibiting pyroptosis of Schwann cells. The morphological features of regenerated nerve fibers can reflect the degree of functional recovery after peripheral nerve injury [45]. In the present study, after treatment with Ac-YVAD-cmk, the average myelinated axon diameter and thickness of the myelin sheath were significantly improved in regenerated nerve fibers after sciatic injury. The SFI value is a reliable index for detecting motor function recovery of injured sciatic nerves. Administration of Ac-YVAD-cmk by intraperitoneal injection in rats caused a noticeable acceleration in the recovery of motor function from the 60th day after surgery. The above evidence proves that Ac-YVAD-cmk can effectively promote peripheral nerve regeneration by attenuating pyroptosis of Schwann cells in rats.

Conclusion
Our studies provide novel insight into one of the mechanisms of Schwann cells in peripheral nerve regeneration. We demonstrate that pyroptosis of Schwann cells induced by peripheral nerve injury impaired peripheral nerve regeneration by releasing inflammatory cytokines, which could be attenuated by the pyroptosis inhibitor Ac-YVAD-cmk. More importantly, inhibition of pyroptosis in Schwann cells may reduce the inflammatory response and promote nerve regeneration in peripheral nerve injury, which provides a potential therapeutic strategy in the future.

Data Availability
All data supporting the findings of this report are included in this article. Source raw data for all quantitations is fully available upon request.

Conflicts of Interest
The authors declare that they have no conflict of interest.

Authors' Contributions
Jiayi Wang, Shunyi Lu, and Ya Yuan contributed equally to this work.  Mediators of Inflammation